The overall objective of this proposal is to identify and characterize specific monovalent metal ion binding sites within RNA tertiary structures. Metal ions act as essential cofactors in the folding of RNAs. The identification of these metal ion binding sites has focused almost exclusively on divalent ions, such as Mg2+, which can play both a structural and a catalytic role within RNAs. While previous research has emphasized divalent ions, many RNAs with essential biological functions explicitly require potassium or sodium cations to fold and/or to promote catalysis in vitro. The ribosome (protein synthesis), the group II intron (intron splicing), and RNase P (tRNA maturation) all require between 0.2 and 2 M monovalent ion to catalyze their respective reactions, and this requirement cannot be overcome by high concentrations of divalent ion. The possibility of specific and functionally relevant monovalent metal ion binding sites within RNA has been largely unexplored, due to a lack of methods with which to identify them. This proposal outlines a new series of biochemical and biophysical approaches to address this problem. These approaches utilize monovalent thallium (Tl+), a heavy metal cation with chemical properties similar to the physiological alkali metals potassium (K+) and sodium (Na+). Tl+ can effectively mimic the biological action of these cations, yet it has properties that will be valuable for the analysis of monovalent metal ion binding to RNA, including excellent NMR receptivity and an enhanced ability to coordinate sulfur. Efforts will be directed toward four research objectives that utilize the chemical properties of TI+ for the study of K+ or Na+ binding sites in RNA. These objectives are: (i) Identification of a K+ metal ion site within all three major classes of large catalytic RNA; (ii) Determination of the structural and biological relevance of a monovalent ion binding site within the ribosomal frameshifting pseudoknot; (iii) Determination the role of two monovalent metal ion binding sites located within the protein-RNA core of the signal recognition particle, one of the most conserved ribonucleoprotein interactions in biology; (iv) Development of 205Tl NMR as a method for the direct detection of monovalent metal ion binding to nucleic acids using the DNA G-quartet as a model system; (iv) Utilization 205Tl NMR to study monovalent metal ion binding in complex RNA systems including catalytic RNAs, pseudoknots, and the signal recognition particle.